Neutrophil-mediated inflammation contributes to the pathogenesis of many diseases including autoinflammatory syndromes, heart disease, asthma, rheumatic disease and inflammatory bowel disease. A key early step in the inflammatory response is the directional migration of neutrophils to sites of inflammation. The overall goal of this proposal is to understand the basic molecular mechanisms that regulate neutrophil chemotaxis in vivo, and to understand how these mechanisms contribute to the development of chronic inflammation using zebrafish as a model system. The strength of this proposal lies in the use of novel transgenic zebrafish lines that we have developed to study neutrophil chemotaxis and inflammation in vivo. Using these tools we have made the observation that zebrafish neutrophils exhibit bidirectional migration between tissues and the vasculature, and reverse neutrophil migration contributes to resolution of the inflammatory response. The hypothesis that guides this research is that neutrophil chemotaxis mediated by G-protein coupled receptors (GPCR), phosphoinositide signaling and the adaptor protein PSTPIP1 modulate bidirectional neutrophil trafficking between tissues and the vasculature and the development of inflammation in vivo. We are now uniquely positioned with improved methods for high efficiency transient expression of transgenes or biosensor probes in zebrafish neutrophils to dissect the molecular mechanisms that regulate neutrophil chemotaxis and bidirectional migration in vivo. Accordingly, we propose the following specific aims: 1. Elucidate how phosphoinositide signaling regulates bidirectional neutrophil trafficking and inflammation in zebrafish. 2. Elucidate how GPCR signaling through CXCR4 regulates bidirectional neutrophil trafficking and inflammation in zebrafish, and 3. Use zebrafish as a model system to examine neutrophil chemotaxis in autoinflammatory disease in vivo. The research proposed in this grant will not only elucidate the molecular mechanisms that regulate neutrophil chemotaxis, but will also likely provide insight into the pathogenesis of chronic inflammation and the development of new therapeutic for control of disease states in which inflammation is central to pathogenesis. PUBLIC HEALTH RELEVANCE: The proposed research contributes to human health by increasing our understanding of the molecular mechanisms that regulate leukocyte motility, wound healing and inflammation using zebrafish as a model system. Knowledge of the basic mechanisms that regulate neutrophil chemotaxis in vivo should aid in the identification of therapeutic targets for autoinflammatory syndromes and other disease states in which inflammation is central to pathogenesis, including heart disease, asthma, arthritis and inflammatory bowel disease.